The overall goal of this project is to elucidate the mechanism of RNA editing in which mRNAs in trypanosome mitochondria are altered by post- transcriptional addition and deletion of uridines. The existing in vitro system will be refined to perform a single cycle of editing at a single site. This system will be used to test the hypotheses that gRNAs specify edited sequences, that uridines which are added or removed by editing are transferred from or to gRNA U-tails, respectively, and that gRNA/mRNA chimeras are editing intermediates, since these issues are central to the mechanism of editing. Studies are also designed to determine whether editing employs transesterification or separate cleavage and ligation reactions. Coordinated physical and functional studies will identify and characterize the macromolecular complex (the editosome) that catalyzes editing; their initial phases will be coordinated with development of the in vitro system. Complexes that occur in vivo and form in vitro and differ between wild type and mutants that do not edit normally will be identified and characterized to determine their interrelationships, role in editing and general assembly sequence. Two gRNA binding proteins of 25 kDa and 90 kDa that are probable components of the editosome will be purified and their genes cloned and characterized. A panel of monoclonal antibodies specific to editosome components will be prepared using the 25 kDa and 90 kDa proteins as initial immunogens and TUTase and editosome complexes that are immunopurified with anti-25 kDa or 90 kDa antibodies as subsequent immunogens. These antibodies will be used to characterize editosome composition, assembly and function. Overall the project is designed to determine critical features of a genetic regulatory process that occurs at the RNA level.